Recently, to deal with exhaustion of fossil fuel and global warming caused by greenhouse gases all over the world, many research and development has been made to produce fuels for transportation and industrial chemicals from renewable biomass. Particularly, bioethanol used as a biofuel is produced by obtaining sugar from biomass, followed by fermentation.
Biomass that is regarded as being reproduced indefinitely so long as the sunlight exists, includes lignocellulosic biomass mainly including plants on the ground and algal biomass mainly including green algae growing in the water. One of the structural components of biomass, cellulose, is the most abundant material on the Earth and occupies 20% to 50% of biomass, and is a product of condensation polymerization from glucose which is the main source of carbon and energy for fermentation strains. Many research and development is being actively made to produce glucose from cellulose with high quantity and quality.
However, in addition to cellulose, lignocellulosic biomass includes hemicellulose (about 15 to 35%) that is susceptible to acid-catalytic hydrolysis and overdegradation, and lignin (about 10 to 30%) difficult to be broken down into monomers having a particular functional group due to its complex structure. Besides, lignocellulosic biomass contains materials that can be extracted with water, such as, for example, water-soluble starch, free sugar, protein, lipid, pectin, tannin, a variety of alkaloids, organic acids and a variety of inorganic salts, and generally, their amounts in herbaceous biomass are large, for example, 20 to 30%, and their amounts in woody biomass are a bit small, for example, 5 to 20% (Michael E. Himmel (2009) Biomass recalcitrance, Blackwell Publishing; Run-Cang Sun (2010) Cereal Straw as a Resource for Sustainable Biomaterials and Biofuels, Elsevier).
Among the extractable materials of lignocellulosic biomass, starch or free sugar can be used in producing fermentable sugar, but materials other than starch or free sugar act as impurities in fermentable sugar and become a factor that causes the sugar yield to reduce in the production of fermentable sugar, so they need to be recovered or removed.
Korean Patent Publication No. 2011-0040367 discloses an apparatus in which as one process of continuous fractionation of biomass, hot water is fed into a reaction tank in which contains biomass, and after stirring for a predetermined time, a liquid is discharged using stream pressure. The above invention was intended to enable fractionation of hot water extractable materials by the apparatus, but liquid discharge through a valve under high pressure is not easy, and due to a tunneling phenomenon within the contents during discharging, even though extraction and recovery is repeated through a valve, the total recovery rate is as extremely low as 50% or less, so the prior art has a technical problem in that it is impossible to use for practical removal of the extractable materials.
It is known that Inbicon, a Danish company specializing in the production of bioethanol with lignocellulosic biomass as a raw material, removes not only extractable materials in biomass but also a liquid containing a large amount of microbial inhibitors by using a process which performs liquid hot water pretreatment of biomass, solid-liquid separation, and subsequent washing the pretreated solid with water (Jan Larsen et al., 2012, Biomass and Bioenergy, 46, 36-45). This method has an advantage in that a clean pretreated solid used to produce a good quality of fermentable sugar can be obtained, but a production cost inevitably increases due to solid-liquid separation and repeated washing. Also, there is a need to perform a process in which a predetermined amount of pretreated liquids containing microbial growth inhibitors should be additionally fed in order to prevent the contamination by lactic acid bacteria during enzymatic hydrolysis or alcohol fermentation, and as a consequence, there is no choice but to add extractable materials of biomass of which composition cannot be known due to a more complex composition caused by a high temperature and pressure reaction. In addition, because a liquid obtained, as a by-product, after pretreatment of biomass contains not only xylose and xylan but also overdegradation products of carbohydrate such as furfural and 5-hydroxymethyl-2-furfural (hereinafter referred to as ‘HMF’), chemically altered proteins produced by the maillard reaction at high temperature of, for example, 190° C., a variety of organic acids, lignin degradation products and a variety of inorganic salts, it is difficult for this by-product to make high value added products but relatively low value added fertilizers.
In producing glucose from lignocellulosic biomass, because only the use of biomass in ground form is not sufficient for converting cellulose to glucose, it is general to perform pretreatment and saccharification processes in a sequential order. The pretreatment of biomass refers to a process which treats ground or crushed biomass by a physicochemical method to bring each structural component of biomass into an easy state to fractionation. During pretreatment of biomass, hemicellulose or lignin surrounding cellulose is degraded or dissolved and released in part or in whole, so that the cellulose becomes more susceptible to be hydrolyzed. The saccharification of biomass refers to conversion of cellulose to glucose by a physicochemical or biochemical method after pretreatment described as the above.
Provided that means for cellulose hydrolysis is limited to enzymes in the saccharification process, the technique being widely used for pretreatment of biomass includes liquid hot water pretreatment (autohydrolysis or hydrothermolysis), dilute acid pretreatment, lime pretreatment, ammonia pretreatment (ARP, etc.), and steam explosion. These pretreatment techniques make cellulose susceptible to hydrolytic enzymes better through a pretreatment by which hemicellulose or lignin in biomass is primarily removed. But, based on the type of biomass and reaction condition, not only the pretreatment efficiency greatly changes, but also the type and amount of materials newly produced other than sugar in the pretreatment or saccharification greatly changes as well. Recently, among these techniques, more attention is paid to liquid hot water pretreatment technique, because it is a simple and most economical process while at the same time having high applicability to a variety of biomasses.
The enzymatic hydrolysis of the pretreatment product refers to a process which converts cellulose to glucose by adding a cellulase formulation to the pretreatment product containing cellulose that is already made more susceptible to enzymes. In this instance, to promote the hydrolysis reaction, the cellulase additionally contains a variety of enzymes such as hemicellulases, starch hydrolase and pectinase in consideration of the pretreatment technique previously applied.
The sugar containing product produced through pretreatment and saccharification of lignocellulosic biomass as a raw material may be used largely for two purposes. First, there is a simultaneous saccharification and co-fermentation method. In this method, fermentation strains and additives are added either directly to a saccharification product in which monosaccharide mainly including glucose is dissolved, but solid residues after hydrolysis (hereinafter referred to as ‘hydrolysis residue’) are also contained (hereinafter referred to as ‘saccharification product’), or to a pretreatment product containing a slight amount of glucose produced by initiation of enzymatic saccharification. Currently, this method is being widely used in research and practical production of bioalcohol. The other methods is that primarily a sugar solution is obtained through solid-liquid separation after saccharification is completed and then uses it as fermentable sugar.
The sugar solution prepared by physicochemical pretreatment and enzymatic hydrolysis of lignocellulosic biomass contains not only monosaccharide including glucose but also many materials as impurities. A typical impurity includes aldehydes produced by overdegradation of sugar such as furfural and HMF, organic acids such as levulinic acid and formic acid, and alcohols such as methanol, and besides, may include acetic acids produced by hydrolysis of hemicellulose and many phenolic compounds produced by degradation of lignin. These impurities may act as microbial growth inhibitors or metabolite production inhibitors depending on the type of fermentation strains. Reportedly, phenolic compounds that are lignin degradation products contained in a sugar solution are commonly the strongest microbial inhibitor, furfural and HMF may serve as a selective inhibitor depending on the concentration, and a variety of acids such as acetic acid may vary in physiological reaction for each strain. When cultivating Clostridium beijerinckii using a sugar solution prepared from corn stover by dilute acid pretreatment using sulfuric acid and enzymatic hydrolysis, furfural and HMF promoted the growth of the strains, while phenolic compounds such as syringaldehyde inhibited the their growth (Thaddeus Ezeji et al, Biothechnology and Bioengineering, 97(6), 1460-1468, 2007). In the study, in which each material was added to an artificially prepared sugar solution at each concentration, and growth was tested using yeast as an ethanologen, furfural did not affect the ethanol production, while HMF had a slight influence, and acetic acid inhibited the growth markedly with the increasing concentration (Jeffrey D. Keating et al, 2006, Biotechnology and Bioengineering, 93(6), 1196-1206). Also, it was reported that phenolic compounds greatly inhibited the growth of yeast, and inhibition performance in the case where acetic acid and furfural were used together was greater than the case where acetic acid and furfural were used singly. In the case of Corynebacterium glutamicum as an ethanologen, the growth was inhibited with the increasing concentration of furfural and HMF, and this growth inhibition was more sensitive to Zymomonas mobilis and E. coli (Shinsuke Sakai et al, Applied and Environmental Microbiology, 2349-2353, 2007). Also, phenolic compounds such as syringaldehyde severely inhibited the growth of the strains, but reportedly, the influence of acetic acid was not great. Such microbial inhibitors contained in a sugar solution produced from lignocellulosic biomass may have different influences on the growth and metabolite production according to the type of microorganisms, so it is not easy to assure a general tendency without any direct application.
Thus, to use the saccharification product produced from biomass intactly for microbial fermentation, attempts have been made to improve fermentation strains molecular biologically or select suitable microorganisms from new strains to avoid the influence by many impurities or efficiently produce metabolite. From a long time ago, an ethanologen or yeast used for humans to produce many types of alcoholic beverages is said to one of fermentation strains having highest resistance to microbial inhibitors, and recently, the intensive studies are being made to make the strains suitable for production of bioalcohol using a lignocellulosic sugar solution. Lactic acid bacteria that is ubiquitous in daily life is also known as a relatively less vulnerable strain.
In contrast, generally, most of industrial microbes including E. coli or Clostridium acetobutylicum are greatly inhibited in growth or metabolite production by several impurities. Thus, for detoxification of many microbial inhibitors contained in a sugar solution prepared from lignocellulosic biomass, many studies have been made, for example, overliming, polymerization using lignin peroxidase, etc. Many other researchers are applying various types of chromatographies using adsorption and partition to removal approach of these materials (Villarreal, M. L. M. et al, Enzyme and Micrbial Technology, 40, 17-24, 2006).
On the other hand, to minimize an amount of microbial inhibitors contained in a sugar solution, a method that washes a pretreatment product with excess water before enzymatic hydrolysis is being used. It is known that Inbicon, now running a pilot plant scale bioalcohol production facility, uses a method which performs liquid hot water pretreatment of biomass, removes a liquid phase of pretreatment product containing a large amount of microbial inhibitors by solid-liquid separation and then washes the pretreated solid with water (Jan Larsen et al, 2012, Biomass and Bioenergy, 46, 36-45). However, it is susceptible to contamination by unwanted microorganisms such as lactic acid bacteria at the initial stage of enzymatic hydrolysis or alcohol fermentation after washing the pretreatment product, therefore, to prevent this, an aliquot of the pretreatment liquid containing microbial growth inhibitors is added back. Also, during enzymatic hydrolysis and ethanol fermentation, microbial inhibitors such as acetic acid and phenolic compounds are released from the pretreatment product, so this method is very useful for alcohol fermentation using yeast having strong resistance to microbial inhibitors, but there is no report on its application to fermentable sugar production for a wide use. Also, in the case of a pretreatment product having a reduction in average diameter by grinding or pretreatment, a portion of the finely particulated pretreatment product may be lost during washing, involving a risk of sugar yield reduction.
Lignocellulosic biomass includes a small amount of extractable materials that can be extracted with water or organic solvents, and structural components that are polymerized and are not dissolved in water or organic solvents. Cellulose is one of structural components of lignocellulosic biomass, occupies 25% to 60% of biomass, and is a polymer made by dehydration condensation of glucose. When producing fermentable sugar using biomass as a raw material, cellulose in biomass is a main target for acid hydrolysis or enzymatic hydrolysis, but because cellulose is surrounded by other structural components, hemicellulose and lignin, cellulose is not easily fractionated.
One of structural components of lignocellulosic biomass, hemicellulose (15 to 35%), has glucose, galactose, mannose and arabinose as side chains linked to the xylan skeleton formed by dehydration condensation of xylose, and acids such as glucuronic acid and acetic acid bonded by ester linkages, and thus, hydrolysis is relatively easy. In contrast, because lignin (10 to 30%) is a polymer having a complex structure of lignan which is an aromatic compound, it is less susceptible to hydrolysis by acids, but is soluble in alkali.
When producing fermentable sugar by enzymatic hydrolysis of cellulose using lignocellulosic biomass as a raw material, to help the action of a cellulolytic enzyme, it is general to first perform physicochemical or biological pretreatment of biomass. The saccharification of biomass by enzymes represents adding cellulase or a mixture of cellulase and hemicellulase to the pretreatment product to hydrolyze cellulose or hemicellulose for a predetermined time to thereby convert to a sugar solution containing glucose and xylose as a primary ingredient.
Liquid hot water pretreatment (autohydrolysis or hydrothermolysis) widely used in pretreatment of herbaceous biomass is a simple technique that puts biomass and water in a high pressure reactor, followed by sealing and causing a reaction at 160° C. to 220° C. for a predetermined time. In this liquid hot water pretreatment, hemicellulose in lignocellulosic biomass is hydrolyzed first and is then released to water by xylooligosaccharides together with xylose, and generally, most of herbaceous and woody biomass shows a maximum yield at 180 to 190° C. Subsequently, the concentration of xylose and xylooligosaccharides detected in water reduces rapidly with the temperature, which is because xylose is degraded further to produce an overdecomposition product such as furfural at a much higher rate than a rate at which hemicellulose in biomass is hydrolyzed and releases xylose to water. However, the concentration of acetic acid in the pretreated liquid gradually increases from 160° C. or lower to 220° C. or higher, and it is thought that most of acetic acid is a hydrolysate of acetyl groups attached to the xylan skeleton of hemicellulose by ester linkages.
Generally, it is said that a drawback of liquid hot water pretreatment is a slightly low sugar yield after enzymatic hydrolysis when compared to dilute acid pretreatment that mainly employs a low concentration of sulfuric acid or hydrochloric acid in pretreatment. In addition, when hemicellulose remaining by imperfect hydrolysis during pretreatment is hydrolyzed by enzymes in a saccharification process, organic acids such as acetic acid are bound to be released, therefore, after enzymatic hydrolysis, such acids are contained in a sugar solution.
The fermentable sugar produced by liquid hot water pretreatment and enzymatic hydrolysis using lignocellulosic biomass as a raw material contains furfural, 5-hydroxymethyl-2-furaldehyde (HMF), phenolic substances and acetic acid as impurities, and these materials are known as inhibiting the microbial growth or metabolite production based on the type of fermentation strains. Reportedly, in the study in which each material was added to an artificially prepared sugar solution at each concentration and growth was tested using yeast as an ethanologen, furfural did not affect the ethanol production, while HMF had a slight influence, but acetic acid inhibited the growth markedly with the increasing concentration (Jeffrey D. Keating et al, 2006, Biotechnology and Bioengineering, 93(6), 1196-1206). Also, it was reported that phenolic compounds greatly inhibited the growth of yeast, and inhibition performance in the case where acetic acid and furfural were used together was greater than the case where acetic acid and furfural were used singly.
Generally, most of industrial microbes including E. coli or Clostridium acetobutylicum are greatly inhibited in growth or metabolite production by several impurities. Thus, for detoxification of many microbial inhibitors contained in a sugar solution prepared from lignocellulosic biomass, many studies have been made, for example, overliming, polymerization using lignin peroxidase, etc. Many other researchers are applying various types of chromatographies using adsorption and partition to removal approach of these materials (Villarreal, M. L. M. et al, Enzyme and Micrbial Technology, 40, 17-24, 2006).
Particularly, many attempts are being made, for example, technology using adsorption chromatography to remove acetic acid contained in a sugar solution (Hee-Geun Nam, Sungyong Mun, 2012, Process Biochemistry, 47, 725-734; S. Ranil Wickramasinghe, David L. Grzenia, 2008, Desalination, 234, 144-151), and technology using a separation membrane (David L. Grzenia et al, 2012, Journal of Membrane Science, 415-415, 75-84; Sung-Jae Kim et al, 2012, Process Biochemistry, 47, 2051-2057].
Overliming for detoxification of a sugar solution is lowest cost and is being widely used, and includes adding and dissolving lime until pH of a sugar solution reaches 10 and heating at 60° C. or less for a predetermined time. As a result, many impurities such as furfural, HMF, protein, etc. may be deposited and removed by filtration or deposition. However, a loss of hemicellulose sugar is generally accompanied during the process.
On the other hand, to minimize an amount of microbial inhibitors contained in a sugar solution, a method that washes a pretreatment product with excess water before enzymatic hydrolysis is being used. It is known that Inbicon, now running a pilot plant scale bioalcohol production facility, uses a method which performs liquid hot water pretreatment of biomass, removes a liquid phase of pretreatment product containing a large amount of microbial inhibitors by solid-liquid separation and then washes the pretreated solid with water (Jan Larsen et al, 2012, Biomass and Bioenergy, 46, 36-45). However, during enzymatic hydrolysis and ethanol fermentation, microbial inhibitors such as acetic acid and phenolic compounds are released from the pretreatment product, and thus, there will be a need for more research and development to apply this method to fermentable sugar production suitable for cultivation of other fermentation strains.
Another method for removing acetic acid from biomass is a method that adds sodium hydroxide to biomass, hydrolyzes acetiyl group in hemicellulose by heating, elutes acetic acid, and carries out solid-liquid separation, and washes out with water to remove acetic acid (Cho, D. H. et al, 2010, Bioresource Technology, 10, 4947-4951). In the case that this method is performed before dilute acid pretreatment of biomass, this method is available because acids are added as a catalyst for pretreatment. That is, biomass having undergone hydrolysis and removal of acetic acid contained in hemicellulose by strong alkali treatment beforehand cannot be expected to undergo a hydrolysis reaction of the hemicellulose main chain by the action of the acid catalyst, so unless acids are artificially added, it should be heated at 230° C. to 250° C. or higher to expect a pretreatment effect. This pretreatment technique is known as pH-controlled liquid hot water pretreatment.
Cellulose that is a structural component of lignocellulosic biomass and a direct raw material for fermentable sugar production occupies 25% to 60% of biomass, and is a polymer made by dehydration condensation of glucose. When producing glucose by acid hydrolysis or enzymatic hydrolysis of cellulose, hemicellulose and lignin included in lignocellulosic biomass acts as a barrier. Thus, before cellulose hydrolysis, pretreatment of biomass for chemically degrading either hemicellulose or lignin or breaking of its rigid structure is essential.
Because hemicellulose (15 to 35%) making up lignocellulosic biomass includes glucose, galactose, mannose and arabinose as side chains linked to the xylan skeleton formed by dehydration condensation of xylose, and organic acids such as uronic acid and acetic acid bonded by ester linkages, hydrolysis by acid catalysts is relatively easy.
Liquid hot water pretreatment (autohydrolysis or hydrothermolysis) or dilute acid treatment widely used for pretreatment of herbaceous biomass to produce fermentable sugar by hydrolyzing biomass using enzymes is technique that puts biomass and water or acids in a high pressure reactor, followed by sealing and causing a reaction at 140° C. to 230° C. for a predetermined time. Hemicellulose in lignocellulosic biomass is hydrolyzed by acid-catalytic pretreatment and released to water by xylooligosaccharides together with xylose, and in this instance, acetyl groups attached to the xylan main chain in hemicellulose by ester linkages are hydrolyzed and released together.
However, the ratio of acetic acid hydrolyzed and released from hemicellulose changes depending on the severity of the pretreatment process, and acetyl groups remaining in unreacted state are hydrolyzed and released in the subsequent enzymatic hydrolysis or acid hydrolysis. To hydrolyze and remove all acetyl groups in hemicellulose during acid catalyst pretreatment, the acid concentration or pretreatment temperature should be increased. However, it is known that at such a high severity, xylose produced by hydrolysis of hemicellulose is overdegraded, yielding 2-furfural, acetic acid and formic acid, and glucose is overdegraded, yielding 5-hydroxymethyl-2-furaldehyde (HMF) and levulinic acid.
In contrast, if the severity of pretreatment is reduced to avoid overdegradation of carbohydrate, when hemicellulose remaining by incomplete hydrolysis in the pretreatment process is hydrolyzed by enzymes during subsequent saccharification, organic acids such as acetic acid are released. Therefore, after enzymatic hydrolysis, such acids are contained in a sugar solution.
The fermentable sugar produced by liquid hot water pretreatment and enzymatic hydrolysis using lignocellulosic biomass as a raw material contains furfural, 5-hydroxymethyl-2-furaldehyde (HMF), phenolic substances and acetic acid as impurities, and these materials are known as inhibiting the microbial growth or metabolite production based on the type of fermentation strains. Reportedly, in the study in which each material was added to an artificially prepared sugar solution at each concentration and growth was tested using yeast as an ethanologen, furfural did not affect the ethanol production, while HMF had a slight influence, but acetic acid inhibited the growth markedly with the increasing concentration (Jeffrey D. Keating et al, 2006, Biotechnology and Bioengineering, 93(6), 1196-1206). Also, it was reported that phenolic compounds greatly inhibited the growth of yeast, and inhibition performance in the case where acetic acid and furfural were used together was greater than the case where acetic acid and furfural were used singly.
Generally, most of industrial microbes including E. coli or Clostridium acetobutylicum are greatly inhibited in growth or metabolite production by several impurities. Thus, for detoxification of many microbial inhibitors contained in a sugar solution prepared from lignocellulosic biomass, many studies have been made, for example, overliming, polymerization using lignin peroxidase, etc. Many other researchers are applying various types of chromatographies using adsorption and partition to removal approach of these materials (Villarreal, M. L. M. et al, Enzyme and Micrbial Technology, 40, 17-24, 2006).
Particularly, many attempts are being made, for example, technology using adsorption chromatography to remove acetic acid contained in a sugar solution (Hee-Geun Nam, Sungyong Mun, 2012, Process Biochemistry, 47, 725-734; S. Ranil Wickramasinghe, David L. Grzenia, 2008, Desalination, 234, 144-151), and technology using a separation membrane (David L. Grzenia et al, 2012, Journal of Membrane Science, 415-415, 75-84; Sung-Jae Kim et al, 2012, Process Biochemistry, 47, 2051-2057].
Overliming for detoxification of a sugar solution is lowest cost and is being widely used, and includes adding and dissolving lime until pH of a sugar solution reaches 10 and heating at 60° C. or less for a predetermined time. As a result, many impurities such as furfural, HMF, protein, etc. may be deposited and removed by filtration or deposition. However, a loss of hemicellulose sugar is generally accompanied during the process.
Another method for removing acetic acid from biomass is a method that adds sodium hydroxide to biomass, hydrolyzes acetiyl group in hemicellulose by heating, elutes acetic acid, and carries out solid-liquid separation, and washes out with water to remove acetic acid (Cho, D. H. et al, 2010, Bioresource Technology, 10, 4947-4951). In the case that this method is performed before dilute acid pretreatment of biomass, this method is available because acids are added as a catalyst for pretreatment. That is, biomass having undergone hydrolysis and removal of acetic acid contained in hemicellulose by strong alkali treatment beforehand cannot be expected to undergo a hydrolysis reaction of the hemicellulose main chain by the action of the acid catalyst, so unless acids are artificially added, it should be heated at 230° C. to 250° C. or higher to expect a pretreatment effect. This pretreatment technique is known as pH-controlled liquid hot water pretreatment.
On the other hand, to minimize an amount of microbial inhibitors contained in a sugar solution, a method that washes a pretreatment product with excess water before enzymatic hydrolysis is being used. It is known that Inbicon, now running a pilot plant scale bioalcohol production facility, uses a method which performs liquid hot water pretreatment of biomass, removes a liquid phase of pretreatment product containing a large amount of microbial inhibitors by solid-liquid separation and then washes the pretreated solid with water (Jan Larsen et al, 2012, Biomass and Bioenergy, 46, 36-45). However, during enzymatic hydrolysis and ethanol fermentation, microbial inhibitors such as acetic acid and phenolic compounds are released from the pretreatment product, and thus, there will be a need for more research and development to apply this method to fermentable sugar production suitable for cultivation of other fermentation strains.
Recently, as a new technique for minimizing an amount of acetic acid produced in the stage of enzymatic hydrolysis, washing a pretreatment product with an aqueous alkaline solution prior to the enzymatic hydrolysis has been suggested (Korean Patent Application No. 10-2013-0082290). But alkali chemicals and excess water are needed, and at least two processes are added, so the cost increase is inevitable in fermentable sugar production.
Lignocellulosic biomass primarily including aboveground plants consists of three kinds of polymers, i.e., cellulose, hemicellulose, and lignin, as structural components forming the structures of the plants, and has many additional materials that can be extracted with water or solvents. Starch that is a storage form of glucose in plants is included in the ‘total glucan’ with cellulose. Glucose made from the total glucan is used as a main carbon source for microorganisms in fermentative production of bioalcohol such as bioethanol or biobuthanol, monomers for biopolymer synthesis such as lactic acid and succinic acid, and metabolite such as acetone and insulin.
Cellulose, one of the structural components of biomass, is not easily converted to glucose by simple acid hydrolysis or enzymatic hydrolysis, because it is densely linked with hemicellulose mainly containing pentoses such as xylose and lignin that is a polymer of phenolic compounds by many chemical bonds, so it is general to additionally hydrolyze with acids or enzymes after pretreatment that usually dissolves either hemicellulose or lignin to expose cellulose.
However, in lignocellulosic biomass containing starch, because relatively and thermochemically stable starch surrounds cellulose together with hemicellulose and lignin, the fractionation of cellulose is more difficult. When converting lignocellulosic biomass containing starch to glucose using ordinary pretreatment and saccharification techniques, pretreatment needs to be performed at higher temperature than lignocellulosic biomass containing no starch to increase the glucose yield. However, at such high temperature, a portion of starch is overdegraded, yielding 5-hydroxy-2-furaldehyde (HMF) which is a microbial growth inhibitor. And starch is first converted to glucose by enzymes during enzymatic hydrolysis, afterward the resulting glucose tends to reduce enzyme activity by feedback inhibition, which makes it difficult to increase the sugar yield.
To overcome this phenomenon, technique that first separates sap from palm trunks containing a large amount of sugar, and converts it to ethanol or lactic acid through fermentation (CN-101589151; JP-2008-178355; Akihiko Kosugi et al, 2010, Ethanol and lactic acid production using sap squeezed from old oil palm trunks felled for replanting, Journal of Bioscience and Bioengineering, 110(3), 322325), and technique that separates parenchyma and vascular bundles from palm trunks and, then, converts them to ethanol (Prawitwong et al, 2012, Efficient ethanol production from separated parenchyma and vascular bundle of oil palm trunk, Bioresour. Technol., 125, 37-42) have ever been reported. Also, a paper investigating ethanol production in which a sugar solution is prepared from ground palm trunks by concentrated sulfuric acid pretreatment, concentrated acid hydrolysis and solid-liquid separation, followed by fermentation by yeast (Chin et al, 2010, Optimization study of ethanolic fermentation from oil palm trunk, rubberwood, and mixed hardwood hydrolysates using Saccharomyces cerevisiae, Bioresour. Technol., 101, 3287-3291), and a paper describing that palm trunks are pretreated with aqueous ammonia, and after solid-liquid separation of the pretreatment product, and only a solid is subjected to enzymatic hydrolysis to obtain a sugar solution, followed by ethanol fermentation of the sugar solution (Jung et al, 2011, Ethanol production from oil palm trunks treated aqueous ammonia and cellulase, Bioresour. Technol., 102, 7307-7312) have ever been reported.
However, to produce a fermentation product such as ethanol or lactic acid as described above, procedural manipulation at many steps is required to efficiently separate and extract starch or sugar from biomass first, so a rise in production cost is inevitable, and in spite of procedural manipulation at many steps, the ethanol yield is not high.
In keeping up with global energy security issues, for example, the climate change issue caused by exhaustion and excessive consumption of fossil fuels and CO2 emission regulation, many countries in the world are dedicated towards developing alternative energy. Thus, attention is being paid to ethanol production using plant biomass such as plant wastes and woody chips with an attempt to develop alternative energy.
Plant biomass mainly includes hemicellulose, cellulose, and lignin. Cellulose is a simple polysaccharide made by dehydration condensation of glucose, and hemicellulose is a complicated polysaccharide made by dehydration condensation of glucose, xylose, mannose, etc. Thus, cellulose and hemicellulose can be converted to sugar by pretreatment technique, for example, hydrolysis, and the sugar can be used as a carbon source to produce biofuel or chemicals fermentatively.
Hydrolysis for converting cellulose or hemicellulose to fermentable sugar includes an enzymatic hydrolysis method using fungus- or bacteria-produced cellulase and a chemical saccharification method using catalysts such as acid and alkali. A typical hydrolysis method includes a concentrated sulfuric acid method, a dilute sulfuric acid method and an enzyme method. The concentrated sulfuric acid method uses higher than 70% of sulfuric acid, and cellulose and hemicellulose is hydrolyzed under the condition of around 70° C. at normal pressure. After hydrolysis, the produced monosaccharide and sulfuric acid are separated, and sulfuric acid is recycled. The concentrated sulfuric acid method is characterized by a high sugar recovery and applicability to various raw materials. The dilute sulfuric acid method is a method that performs hydrolysis using sulfuric acid in concentration of a few % under the condition of temperature of 150˜250° C. and pressure of 1˜2 MPa. In this instance, because dilute sulfuric acid is used, it is general to perform neutralization treatment without recycling sulfuric acid. Because the dilute sulfuric acid method does not recover or reuse sulfuric acid, the process configuration is simple, but due to the high temperature and high pressure condition, sugar is susceptible to overdegradation, therefore, a drawback is that a recovery rate of monosaccharides is not high. The enzyme method is a method that performs hydrolysis using enzymes. Because this method should bring enzymes into contact with cellulose or hemicellulose efficiently, biomass needs to be degraded to some extent beforehand using dilute sulfuric acid or vapor. Also, there is a need for development to prepare a special enzyme for efficiently breaking the strong linkages between each structural components using genetic modification technology. A primary facility of the enzyme method only involves mixing of enzymes and biomass in a tank, so a low facility cost is an advantage, but a high production cost of enzymes is a disadvantage.
In hydrolysis of cellulose-containing biomass, cellulose or hemicellulose are degraded, while at the same time, producing by-products, for example, furan compounds such as furfural, hydroxymethylfurfural, etc., or organic acids such as formic acid, acetic acid, levulinic acid, etc. Also, because cellulose-containing biomass contains lignin that are aromatic polymers, during acid pretreatment, lignin substances are degraded, and by-products, for example, aromatic compounds such as low molecular weight phenolic compounds are produced. These compounds inhibitively acts on a fermentation process using microorganisms to cause microbial growth inhibition, and reduce the yield of fermentation products, so they are called fermentation inhibitors, and need to be removed when a biomass derived sugar solution is used as a fermentation raw material.
As a conventional method of removing fermentation inhibitors in a preparation process of a sugar solution, Korean Patent Publication No. 2011-94005 discloses a method for preparing a sugar solution, including a process of hydrolyzing cellulose-containing biomass to prepare an aqueous sugar solution; and a process of filtering the obtained aqueous sugar solution through a nanofiltration membrane and/or reverse osmosis membrane to recover a refined sugar solution from the non-permeate side and remove fermentation inhibitors from the permeate side. However, the method is easy to separate fermentation inhibitors but is undesirable in that monosaccharides such as glucose, xylose, etc., are released together.
In this context, the inventors discovered that when a polyamide nanofiltration membrane is modified to reduce surface charge, and an aqueous sugar solution is filtered using the modified polyamide nanofiltration membrane, fermentation inhibitors can be removed and a refined sugar solution is able to be prepared, and thereby, completed the invention.